Medium for analytic and preparative electrophoresis

ABSTRACT

The invention relates to a medium for analytic and preparative electrophoresis comprising a content of acids and bases of different pK S  values. Said medium contains at least two acids, whose pK S  values (ΔpK S ) of adjacent acids are no greater than 1.0 but preferably range from 0.8 to 0.5, and contains at least two bases, whose pK S  values range from approximately 1.5 to 11, whereby the difference of the pK S  values (ΔpK S ) of adjacent bases is no greater than 1.0 but preferably ranges from 0.8 to 0.5.

[0001] This present invention concerns a medium for analytical and preparative electrophoresis.

[0002] Such aqueous solutions, which form a pH gradient upon high-voltage electrophoresis, are used on a large scale to analyze proteins and peptides. Such prior art solutions contain polyelectrolytes with amphoteric properties and are usually manufactured synthetically by means of condensation and/or polymerization of polyamines with unsaturated carboxylic acids. As a result of such synthesis, many different compounds with different molecular weights and a varying number of acid groups (carboxylic groups) and basic groups (amino and/or imino groups) are produced. Under the conditions prevalent during high-voltage electrophoresis, these polyelectrolytes arrange themselves, due to the different ratio between acidic and basic groups, in such a way that polyelectrolytes with particularly acidic properties concentrate in the proximity of the anode, and all other polyelectrolytes are arranged, in accordance with the “ranking” of acidic and basic properties, between the electrodes and also concentrated in the process. By the selective accumulation of polyelectrolytes in accordance with the acidic and/or basic properties of the individual species, a pH gradient is produced throughout the entire aqueous solution.

[0003] By means of this pH gradient, amphoteric compounds such as proteins and peptides can be separated in accordance with the different acidic and/or basic properties. This analytical separation procedure is referred to in the literature as “isoelectric focusing”. In case this procedure is carried out in a special gel (polyacrylamide), this procedure is referred to as PAGIEF (polyacrylamide isoelectric focusing). PAGIEF is considered the standard procedure.

[0004] The unsaturated acids that are used in the synthesis of commercially available ampholytes, e.g. substituted acrylic acids, are highly toxic. Their residual content is considered to represent a significant hazard.

[0005] For that reason, the use of commercially available ampholytes for the cleaning of proteins for subsequent use in human applications is prohibited.

[0006] During ampholyte synthesis, a large number of chemical species of unknown structure is formed whose relative concentration may vary significantly across batches. This has an adverse impact on the reproducibility of analytical results.

[0007] In certain applications, “artificial bands” are reported as a result of undesired complex formation between the ampholyte and the analyte.

[0008] Partial interference with the analytical color reaction of proteins caused by ampholytes with a higher molecular weight has also been reported. An unusually high percentage of ampholytes with a molecular weight>10 KD in products of certain manufacturers has been reported on several occasions.

[0009] In the case of preparative cleaning of proteins, the subsequent separation of the ampholyte is difficult and sometimes incomplete; the suspected cause is complex formation.

[0010] Commercial ampholytes have been found to be unsuitable for use as separating media for bioparticles, considering that bioparticles aggregate in these media.

[0011] The object of this invention is to provide aqueous solutions for isoelectric focusing (IF) which contain acids and bases with different pK_(S) values which are not subject to the above-mentioned disadvantages and whose range of applications is therefore significantly wider.

[0012] The object of this present invention is achieved by the aqueous solution in accordance with claim 1. Other advantageous embodiments thereof are described in the subsequent claims 2 through 7.

[0013] The aqueous solution in accordance with this invention contains a mixture of acids and bases with graded “acidity” and/or “basicity” levels. The pK_(S) values of the individual acids vary between pK_(S) 3.5 to pK_(S) 12, and the differences of the pK_(S) values (ΔpK_(S)) of the “neighboring” acids should not exceed ΔpK_(S)=1. A value of ΔpK_(S) of less than 0.8 is considered ideal.

[0014] The pK_(S) values of the bases used vary between pK_(S)=1.5 and pK_(S)=11. The differences between the values of the individual bases are ideal in case ΔpK_(S)<0.8 is fulfilled.

[0015] The number of acids and/or bases used and their relative concentrations determine the range and the profile of the pH gradient. The minimum number is 2 acids and/or 2 bases each, and 3 acids and/or 3 bases are preferred. In the case of a wide pH range (pH 2.5 to 10), 11 acids and 13 bases with graded pK_(S) values are used.

[0016]FIGS. 1 through 3, attached hereto, use a model of three typical acids and bases each to depict the electrophoretic migration and the resulting establishment of the pH gradient. Different concentrations of the acids hydroxyl butyric acid (HIBA: pK_(S)=3.8), HEPES (pK_(S)=7.5), and 2-hydroxy pyridine (pK_(S)=11.6), and the bases taurine (pK_(S)=1.5), melamine (pK_(S)=5), and ammediol (pK_(S)=9) are contained in the original solution (see FIG. 1). As a general rule, the relative concentrations of both very weak acids and very weak bases are significantly higher compared with the concentrations of the relatively strong acids and bases (approx. by a factor of five). The concentrations of acids and basis of “moderate” strength are increased by a factor of approx. 1.5 to a maximum of 3.

[0017]FIGS. 2 and 3 show the relative concentrations of the above-described acids and bases after electrophoretic migration. In the “state of equilibrium” shown herein, “pairs” of acids and bases are formed which may only ionize each other to a very limited degree (strong acid: HIBA and very weak base: taurine, weak acid: HEPES and weak base: melamine, and strong base: ammediol and very weak acid: 2OH pyridine) , and as a result of thereof, the electrophoretic migration of the media's own substances comes to a stop. In the area of these pairs, a pH value is produced which corresponds to the arithmetic mean of the pK_(S) values of the acid and of the base. The pH values of these pairs are therefore as follows: HIBA/taurine solution, pH=2.6; HEPES/melamine, pH=6.25; 2-OH pyridine/ammediol, pH=10.3. The pH range over which a pair is able to ensure a stable pH value is approx. one pH unit. All other acids and bases with different pK_(S) values form pairs which also ensure a stable pH value within one pH unit as long as the absolute concentrations of the individual acids and bases guarantee sufficient buffer capacity in the state of equilibrium.

[0018] The solutions in accordance with this invention consist of well-defined chemicals whose properties are known in detail. The reproducibility of the entire mixture is ensured by the precision and/or accuracy of the scale used in its production. The substances used are non-toxic. Each of the substances used is “monofunctional”, i.e. effects such as complex formation and/or biopolymer aggregation do either not occur at all or are at least significantly reduced compared with polyelectrolytes comprising several functional groups. The molecular weight of all individual species of the mixture is <300 Dalton.

[0019] The aqueous solutions in accordance with this invention have already been successfully tested as separating media for biopolymers and bioparticles and have been found to be biocompatible. Considering that most individual species can be substituted by chemicals with similar pK_(S) values, the special requirements of bioparticles in terms of separating media can be taken into account by changing the composition of the mixture. For the above reasons, official approval of such solutions for use in the cleaning of bioparticles and biopolymers (peptides and proteins) for subsequent application in humans appears possible.

[0020] Below, several examples of aqueous solutions in accordance with this invention with different pH intervals are presented. Please note that, during the manufacture of a medium for a narrower pH interval, the absolute concentrations of acids and bases are increased; usually, the differences in the pK_(S) values are also reduced by using new constituents.

EXAMPLE 1 (pH Interval 3-10)

[0021] c c Acid pK_(s) (mMol) Base pK_(s) (mMol) Hydroxy 4.0 1.5 Betaine 1.8 10 Isobutyric Acid Pivalic Acid 5.0 5.0 2-Amino 2.3 10 Butyric Acid Picolinic Acid 5.5 2 Creatine 2.7 10 MES 6.1 4 Nicotinic 3.1 8 Acid Amide MOPSO 6.9 2 Pip-4-Carboxylic 3.5 3 Acid HEPES 7.5 2 EACA 4.4 2 EPPS 8.0 2 Melamine 5.0 2 TAPS 8.3 3 Hydroxyethyl 5.3 2 Pyridine Histidine 6.0 3 AMPSO 9.0 6 BISTRIS 6.5 3 CAPSO 9.6 8 Hydroxyethl 7.1 3 Morpholine CAPS 10.3 9 Triethanol 7.8 2 Amine CABS 10.8 10 TRIS 8.3 2 2-Hydroxy-Pyridine 11.8 10 Ammediol 9.0 2

EXAMPLE 2 (pH Interval 4-6)

[0022] Acid pK_(s) c (mMol) Base pK_(s) c (mMol) Pivalic Acid 5.0 5 Creatine 2.7 30 Glycl Picolinic Acid 5.5 10 Glycine 3.1 20 MES 6.1 20 Pip.-4- 3.5 10 Carboxylic Acid MOPSO 6.9 30 GABA 4.2 10 EACA 4.4 5

EXAMPLE 3 (pH Interval 5.5-7.5)

[0023] Acid pK_(s) c (mMol) Base pK_(s) c (mMol) MOPSO 6.9 10 EACA 4.4 15 MOPS 7.2 10 Creatinine 4.8 15 HEPES 7.5 15 Hydroxyethyl 5.4 10 Pyridine EPPS 8.0 15 β-Picoline 5.8 10 TAPS 8.3 20 BISTRIS 6.5 5

EXAMPLE 4 (pH Interval 8-11.5)

[0024] Acid pK_(s) C (mMol) Base pK_(s) c (mMol) TAPS 8.3 3 Imidazol 7.0 15 Triethanol AMPSO 9.0 5 Amine 7.8 12.5 CAPSO 9.6 7.5 TRIS 8.3 12.5 GABA 10.2 10 Ammediol 8.8 10 Hydroxy Ethyl EACA 10.6 10 Ethylene 9.8 7.5 Diamine Diethyl Amino 4-Hydroxy- 11.2 7.5 Ethylene 10.2 n5 Pyridine Diamine 2-Hydroxy- 11.6 7.5 Triethyl 10.8 5 Pyridine Amine Dimethyl 11.2 3 Piperidine

EXAMPLE 5 (pH Interval 2.5-5)

[0025] Acid pK_(s) c (mMol) Base pK_(s) c (mMol) Citric Acid 3.2 5 Betaine 1.8 20 HIBA 4.0 10 2-Amino 2.2 20 Butyric Acid Isobutyric Acid 4.5 15 Creatine 2.7 15 Glycyl Picolinic Acid 5.5 20 Glycine 3.1 10 MES 6.1 20 Pip.-4 3.6 10 Carboxylic Acid GABA 4.2 5

[0026] In the specified media, mostly linear pH gradients are produced. The pH profile can be flattened in certain pH segments by modifying the relative concentrations of acids and bases. In the manufacture of the solutions in accordance with this invention, the order in which the individual compounds are added is usually irrelevant. 

1. A medium for analytical and preparative electrophoresis containing acids and bases with different pK_(S) values, characterized in that it contains at least two acids whose pK_(S) values are between approx. 3.0 and 12, wherein the difference of the pK_(S) values (ΔpK_(S)) of neighboring acids is not larger than 1 and preferably between 0.8 and 0.5, and at least two bases whose pK_(S) values are between approx. 1.5 and 11, wherein the difference between the pK_(S) values (ΔpK_(S)) of neighboring bases is not larger than 1 and preferably between 0.8 and 0.5.
 2. The medium in accordance with claim 1, characterized in that it contains at least three acids and three bases.
 3. The medium in accordance with claim 1 or 2, characterized in that the relative and absolute concentrations of the acids continuously increase with increasing pK_(S) values and the relative and absolute concentrations of the bases decrease with increasing pK_(S) values, wherein, always compared with the concentrations of the strongest acids and bases, the concentrations of the weaker acids and/or weaker bases, in the case of small differences between the pK_(S) values in the range from 1 to 3, are increased by a factor of 1.5 to 3, whereas in the case of large differences between the pK_(S) values in the range from 3 to 8, the relative concentrations of the weaker acids and bases are increased by up to a factor of 4 to
 8. 4. The medium in accordance with any one of the preceding claims, characterized in that, for each neighboring pH value unit, upon completion of electrophoretic migration, an acid/base pair is formed which produces a pH value which corresponds to the arithmetic mean of the pK_(S) values of the respective acid and of the respective base.
 5. The medium in accordance with any one of the preceding claims, characterized in that the concentrations of the individual acids and bases are selected to establish and ensure sufficient buffer capacity in the state or equilibrium upon completion of the electrophoretic migration.
 6. The medium in accordance with any one of the preceding claims, characterized in that, in order to establish narrower pH intervals, the absolute concentrations of the acids and bases and, optionally, the ΔpK_(S) values of the acids and bases are reduced through appropriate selection of the acids and bases.
 7. The medium in accordance with any one of the preceding claims, characterized in that the molecular weights of both the acids and/or bases used are less than 300 Dalton. 